The long-term objective of this project is to delineate mechanisms of vascular calcification in patients with chronic kidney disease-mineral bone disorder (CKD-MBD). Vascular calcification is a major killer of CKD-MBD patients primarily through the heightened risk of cardiovascular morbidity and mortality. Elevated phosphate at levels of hyperphosphatemia has been identified as a key inducer of vascular calcification via procalcific effects on vascular smooth muscle cells (VSMC). In the previous funding period, we identified a novel function for the sodium dependent phosphate transporter, PiT-1, as a mediator of elevated-phosphate-induced vascular calcification in vitro and in vivo. Moreover, we provide mechanistic insight into compensatory mechanisms of the alternative family member, PiT-2, that operates in VSMC to protect against phosphate transporter deficiency. Importantly, as our studies were in progress, PiT-2 was identified as the causative gene for idiopathic basal ganglion calcification in people, and thus our studies noting compensatory mechanisms for phosphate transporters may help to explain how mutation of PiT-2 might lead to compensatory changes that actually facilitate vascular calcification. Clinically, our data provide a cautionary note on compensatory pathways that should be considered when attempting to translate inhibition of phosphate transport to clinical therapies. In addition to the Pi transport through PiTs, our preliminary findings also suggest a Pi transport- independent Pi sensing and signaling mechanism that mediates Pi-driving cell functions. PiT-1 and PiT-2 are high affinity, low capacity Pi transporters that form oligomers under low to normal extracellular Pi, leading to a influx of Pi as source for loading into matrix vesicles that mediate matrix calcification. Under high extracellular Pi, such as hyperphosphatemia in CKD, monomers form, resulting in exposure of cryptic site that binds to PiT adaptor proteins and thereby facilitates Erk1/2 phosphorylation and subsequent signaling to osteochondrogenic differentiation, autophagy, and apoptosis of VSMC. Therefore, in specific aim 1 of the proposed project we will improve scientific knowledge by delineating unique mechanisms responsible for vascular calcification in CKD-MBD and thereby uncover new therapeutic targets and drug targeting modalities that can treat vascular calcification and spare bone. In specific aim 2, we will explore the novel concept of how Pi transport-independent Pi sensing and signaling process is controlled by differential oligomerization that induces VSMC osteochondrogenic phenotype change, apoptosis and autophagy. The proposed work will improve our understanding of how elevated Pi promotes vascular calcification and provide additional therapeutic targets for preventing or treating CKD-MBD.